A common variant in PIK3CG gene associated with the prognosis of heart failure

Abstract Phosphoinositide 3‐kinase γ (PI3Kγ) is G‐protein‐coupled receptor‐activated lipid kinase with both kinase‐dependent and kinase‐independent activity. Plenty of evidence have demonstrated that PI3Kγ participated in TAC and I/R‐induced myocardial remodelling and heart failure (HF). In this study, we tested the hypothesis that common variants in the PI3Kγ gene (PIK3CG) were associated with the prognosis of HF in the Chinese Han population. Through re‐sequencing and genotyping, we finally identified a common variant in the 3′UTR of PIK3CG strongly associated with the prognosis of HF in two‐stage population: adjusted p = 0.007, hazard ratio = 0.56 (0.36–0.85) in the first cohort and adjusted p = 0.024, hazard ratio = 0.39 (0.17–0.88) in the replicated cohort. A series of functional assays revealed that rs10215499‐A allele suppressed PIK3CG translation by facilitating has‐miR‐133a‐3p binding, but not the G allele. Subjects carrying the GG genotype showed higher mRNA and protein level than those with AA and AG genotype. Furthermore, overexpression of PIK3CG could protect AC16 from hypoxia/reoxygenation (H/R)‐induced apoptosis, while the case was opposite for PIK3CG silencing. In conclusion, common variant rs10215499 in the 3′‐UTR of PIK3CG might affect the prognosis of HF by interfering with miR‐133a‐3p binding and PIK3CG is a promising target for HF treatment in the future.

of phosphatidylinositol (PtdIns) (4,5) P2 could be phosphorylated by active PI3K and converted into phosphatidylinositol (3,4,5)-trisphosphate (PIP3), 10 followed by activation of a series of downstream targets including Akt/protein kinase B (PKB). 11cording to the structure, function and substrate specificity, PI3Ks are divided into three different classes. 12A total of four different class I PI3Ks have been discovered, among which PI3Kα, PI3Kβ and PI3Kδ (class I A ) isoenzymes are activated by receptor tyrosine kinase pathway. 9As a single member of Class I B , PI3Kγ heterodimerizes with a regulatory p87 or p101 subunit and is activated by βγ subunit of G-proteins and functions downstream of G protein-coupled receptors (GPCRs). 13,14[18] Substantial evidence has demonstrated that PI3Kγ is involved in the internalization of activated β-adrenergic receptor via its kinase activity. 19,202][23] Importantly, both the protein level and lipid kinase activity of PI3Kγ have been shown to be significantly upregulated in heart failure, 18,19,24 suggesting a vital role of PI3Kγ in the pathological process of HF.Indeed, several reports have demonstrated that the PI3Kγ-selective inhibitor AS605240 could ameliorate cardiac remodelling and dysfunction in TAC-and MI-induced mouse HF models, 21,25 which indicated the deleterious role of kinase-dependent function in myocardium.However, a study by Mauro et al. found that AS605240 significantly worsened MI-induced cardiac dysfunction compared to control. 9On the contrary, the kinase-independent scaffolding function of PI3Kγ was doubtlessly cardio-protective as knock-in for a catalytically inactive PI3Kγ (PI3Kγ-KD) renders animals resistant to cardiac dysfunction under pathological conditions. 10,19,21,22Overall, PI3Kγ consistently displayed favourable role in various HF models. 10,11,22 to now, numerous genetic studies have examined the correlation between variants in the PI3Kγ gene PIK3CG and various disease states, including autistic disorder, 26 subclinical atherosclerosis traits, 27 platelet aggregation, 28 poor responsiveness to clopidogrel, 29 and others.Considering the importance of PI3Kγ in cardiac pathophysiology and the lack of relevant genetic investigations in HF, we hypothesize that PIK3CG polymorphism may serve as a potential prognostic indicator for HF.

| Study population
In the first cohort, a total of 2267 HF patients were successfully recruited for this study between 1 January 2009 and 31 October 2014 in Cardiology.
Division of Tongji Hospital in Wuhan.In the replicated cohort, 838 patients with chronic HF were used to validate the results observed in the first cohort.We defined cardiovascular deaths or cardiac transplantation as the primary end point.Details regarding the diagnostic and exclusion criteria for HF, data collection, and definition of risk factors have been previously described. 4The clinical characteristics of the study populations were summarized in Table 1.
All protocols and methods were approved by the ethics committees of Tongji Hospital and conducted in accordance with the Declaration of Helsinki.We obtained written informed consents from all participants.

| Genetic variation screening
Genomic DNA was extracted from peripheral venous blood samples obtained from 48 control participants, as previously reported. 30Sanger sequencing was performed to identify common variants in the PIK3CG gene.The promoter region and exons region in PIK3CG were amplified using polymerase-chain-reaction (PCR) and subsequently subjected to fluorescent dye-terminator cycle sequencing.The primer sequences used for amplification can be found in Table S1.

| Genotyping
Probe and primer sequences were designed using ABI Primer Expression 3.0 software and synthesized by Shanghai GeneCore Bio Technologies Co., Ltd, China.Genomic DNA was extracted from peripheral leucocytes as previously reported. 4The selected variants in PIK3CG gene were genotyped using the TaqMan assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster TA B L E 1 Baseline characteristics of the study population.City, CA).Detailed procedures and conditions have been described previously. 31The probe and primer sequences are listed in Table S2.
The rs1129293 single nucleotide polymorphism (SNP) was introduced into this expression vector using Fast Mutagenesis System (Beijing TransGen Biotech Co., Ltd.) according to the manufacturer's instructions.Additionally, approximately 400 nucleotides located around rs3173908, rs10216210, rs10215499 and rs6150272 were respectively cloned into pMIR-Report Luciferase.Detailed primer sequences were listed in Table S3.Subsequently, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with a peroxidaseconjugated secondary antibody.Bands were visualized by enhanced chemiluminescence reagents (Pierce Chemical, Rockford, IL) and quantified by densitometry.

| Levels in human heart samples
To assess the effect of functional variant on the PIK3CG expression, we collected 194 samples of peripheral blood lymphocytes from participants undergoing coronary angiography in total.Details about RNA isolation, mRNA transcription, and PCR conducted in this study have been described previously. 30Absolute quantification methods were used to measure the mRNA levels of PIK3CG and ACTB with each sample in triplicate.Relevant primer sequences and detailed characteristics of individuals are shown in Tables S4 and S5, respectively.Expression of PIK3CG relative to ACTB was compared between individuals with TT genotype and with CC or CT genotype.
In addition, a total of 17 human heart samples were used to examine PIK3CG protein expression.The study was approved by the Review Board of Tongji College of Medicine and conducted in accordance with the principles of the Helsinki Declaration.Written informed consents were obtained from all patients.Detailed clinical characteristics of patients are listed in Table S6.

| Determination of hsa-miR-133a-3p expression levels
The expression of hsa-miR-133a-3p was measured using quantitative RT-PCR.Human normal tissues including heart, adrenal gland, small intestine, adipose, skin, muscle, lung and large intestine were obtained from distal normal tissue of tumour patients.Detailed procedures for RNA extraction and quantitative RT-PCR have been described previously. 33The study was approved by the Review Board of Tongji College of Medicine and conducted in accordance with the principles of the Helsinki Declaration.Written informed consents were obtained from all patients.

| Analysis of cell apoptosis
The impact of PIK3CG on H/R-induced apoptosis was assessed by terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining using a TUNEL fluorescence kit (Beyotime Biotechnology).Briefly, AC16 cells were firstly transfected with siRNAs (100 nm/L) or a PIK3CG-overexpression plasmid in a six-well plate and then re-plated in a 24-well plate 24 h later, followed by H/R treatment.For H/R induction, AC16 cells were cultivated in serumfree DMEM in a hypoxia chamber with 5% CO 2 and 95% N 2 for 30 min, then reoxygenated in a humidified atmosphere with 5% CO 2 and 95% air at 37°C for 1 h in DMEM supplemented with 10% FBS.
Following 24 h of H/R treatment, all cells were subjected to TUNEL analysis following standard protocols.The stained apoptotic cells were visualized under the inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

| MATERIAL S
Anti-PIK3CG and anti-ACTB antibody were purchased from Cell Signalling Technology (#5405) and ABclonal Technology (AC026), respectively.The sequence of siRNAs targeting PIK3CG gene was as follows: CTGGCATTTTAGATACGAA, which was designed and synthesized by Guangzhou RiboBio Co., Ltd.TUNEL fluorescence kit was from Beyotime Biotechnology (C1088).(Figure 1A).All the variants were found to be in Hardy-Weinberg equilibrium in our population (p > 0.05).Considering a substantial of variants in PIK3CG, we attempt to narrow down the candidate SNPs with MAF >0.1.Subsequently, we conducted linkage analysis and identified three haplotype structures (Table 3).

| Association of tagged SNPs with the prognosis of HF
Firstly, we selected rs1129293, rs17847825 and rs12667819 as the tagged SNPs for genotyping from the corresponding haplotype.As shown in With major allele given first, followed by minor allele.

TA B L E 2 Characteristics of PIK3CG
variants identified by Sanger sequencing.
were consistent, with the rs1129293-C allele being significantly associated with an increased risk of cardiovascular death and cardiac transplantation (adjusted p = 0.024, HR = 2.59) (Table 4, Figure 2B).Finally, we combined the two populations, and the results indicated that the rs1129293-T allele was significantly associated with a more favourable prognosis compared to the C allele (Table 4, Figure 2C).

| SNP rs1129293 is associated with increase in PIK3CG expression
We then investigated the mechanism underlying the reduction in HF-related mortality risk mediated by the variant rs1129293.
Since rs1129293 is synonymous and 4 other SNPs in strong linkage disequilibrium with rs1129293 were all located in the 3′UTR, we attempted to explore whether the variant could affect PIK3CG expression.Firstly, we compared the PIK3CG mRNA relative expression in lymphocytes, including 194 samples (85 CC, 83 CT and 26 TT genotype).The result revealed that PIK3CG transcription was increased in the samples from individuals with the TT genotype compared with those with the CC and CT genotypes (Figure 3A), suggesting that the variant affects PIK3CG expression.Subsequently, we examined PIK3CG protein expression in 17 human heart tissues with different genotypes using western blotting.The results indicated that PIK3CG protein levels were higher in tissues with TT genotype compared with those with CC and CT genotypes (Figure 3B).

| Association between rs1129293 and parameters of cardiac ultrasonography
Furthermore, we compared the cardiac ultrasound parameters among patients with different genotypes.As shown in Table 5, no significant differences were observed in terms of left ventricular

| Functional analysis of the cause SNP
Considering the possibility that synonymous variants could also disturb the protein expression of corresponding gene, 34 we attempt to explore the effect of rs1129293 on PIK3CG protein level.In AC16 and HEK293T cells, there was no difference in the expression level of PIK3CG between the rs1129293-C and T alleles (Figure 1B,C).
Subsequently, we performed luciferase assay to assess the remaining variants in the 3′UTR, which were in strict linkage disequilibrium with rs1129293.As shown in Figure 4A, the expression of the reporter gene with the rs10215499-G allele significantly increased compared to the rs10215499-A allele, and this finding was replicated in HEK293T cells (Figure S1).However, we did not observe any effect on luciferase activity in rs3173908, rs10216210 and rs6150272 luciferase assays in AC16 cells (Figure 4A).
These results suggest that rs10215499 may be the causal SNP responsible for the different prognosis of HF.
To further validate the effects of hsa-miR-133a-3p on endogenous PIK3CG expression, we sequenced AC16 and HEK293T cell lines and found them to be rs10215499-AA genotype (Figure 5A,B).
Western blot results demonstrated that miR-133a-3p downregulated PIK3CG expression, and the inhibition of miR-133a-3p expression using its inhibitor significantly upregulated expression of PIK3CG in AC16 and HEK293T cells (Figure 5C,D).These findings collectively suggest that the rs10215499-G allele may disrupt the binding site of hsa-miR-133a-3p and consequently increase the expression of PIK3CG, ultimately contributing to improved cardioprotection and prognosis for patients with heart failure.

| Heart hsa-miR-133a-3p level
Real-time quantitative PCR was performed for measurement of hsa- hsa-miR-133a-3p was consistent across heart tissues with different genotypes (Figure 6A).Additionally, hsa-miR-133a-3p was found to be expressed in various human tissues, with the highest expression level observed in the heart (Figure 6B).

| Apoptosis assay
The biological function of PIK3CG under H/R conditions was investigated by overexpressing and silencing PIK3CG in AC16 cells.
Firstly, the pcDNA3.1-PIK3CGvector and PIK3CG-siRNA could significantly increase and reduce the expression level of PIK3CG, respectively (Figure 7A,B).Following 24 h of H/R treatment, AC16 cells with PIK3CG overexpression exhibited a lower apoptosis rate compared to the control group (Figure 7C).Conversely, H/Rinduced apoptosis in AC16 cells was aggravated by PIK3CG-siRNA transfection (Figure 7C).These findings demonstrated a protective role of PIK3CG in H/R-induced apoptosis in cardiomyocytes in vitro.

| DISCUSS ION
Heart failure is a complex clinical syndrome with pathophysiological heterogeneity. 35There is substantial evidence suggesting the involvement of PI3Kγ in the pathological processes of heart failure. 9,10In this study, we identified a common variant rs10215499 in the 3′UTR of PIK3CG gene associated with the prognosis of HF in both the first cohort (p = 0.013, HR = 0.56) and replicated cohort (p = 0.032, HR = 0.39).Importantly, this association remains significant even after adjusting for traditional risk factors, including age, gender, hypertension, diabetes, hyperlipidemia, smoking status, and the use of β-blockers.
As a catalytic subunit, PI3Kγ formed a heterodimer with p87 or p101 subunit and has been extensively studied in various physiological systems, including immune, cardiovascular, endocrine, neuronal functions, as well as its role in malignancy. 36Importantly, the function of PI3Kγ in the myocardium has gained considerable attention due to its abundant expression in cardiovascular tissue. 9merous studies have demonstrated the protective effects of PI3Kγ against myocardial injury and remodelling induced by I/R and TAC, with these effects being abolished in PI3Kγ knockout mice. 9,10,22Our in vitro investigation also demonstrated that PI3Kγ confers resistance to H/R-induced apoptosis in AC16 cells, consistent with the findings of Mauro et al., who reported that PI3Kγ knockout significantly increased cardiomyocyte apoptosis in a mouse model of myocardial infarction (MI). 9Furthermore, increased expression levels and activity of PI3Kγ were observed in the myocardium of mice with TAC-induced heart failure and in patients with heart failure. 18,22Considering the important role of  TA B L E 5 Cardiac ultrasound parameters grouped by rs1129293.

F I G U R E 4
The G allele of rs10215499 destroys a binding site for microRNAs.(A) Luciferase assays showed that only rs10215499 displayed different transcriptional activity between wild-type and mutant-type alleles in AC16 cells.(B) Bioinformatic analyses showed that the allele G of rs10215499 destroyed a binding site for has-miR-133a-3p, has-miR-133b, has-miR-4259 and has-miR-4715-5p.(C) Luciferase assays showed that hsa-miR-133a-3p displayed rs10215499-A allele-dependent inhibiting effect on the luciferase activity.While similar results were not observed for has-miR-4259, has-miR-133b and has-miR-4715-5p (D).NS, not significant; *p < 0.05.
we identified a common SNP located in the 3′UTR that displayed strong association with the prognosis of HF in both first (adjusted p = 0.007, HR = 1.80) and replicated (adjusted p = 0.024, HR = 2.59) cohorts.Importantly, this association was independent of current known risk factors as the statistical significance remained after adjustment for sex, age, hypertension, diabetes, hyperlipidemia, smoking state, and β-blocker use.
To date, numerous genetic investigations have been conducted on the PIK3CG gene.For instance, rs4288294 and rs116697954 in PIK3CG have been reported to be associated with plasma HDLcholesterol concentrations. 15In a case-control study, Gu et al.
identified an association between rs12667819 in 3′UTR of PIK3CG and Attention-deficit/hyperactivity disorder (ADHD). 37Additionally, a positive correlation between PIK3CG SNPs (rs1129293 and rs17398575) and patients with poor responsiveness to clopidogrel may exist. 29However, no investigation has been conducted to explore the role of genetic variants in PIK3CG in HF.Our study was the first to demonstrate the association of variant in PIK3CG with the prognosis of HF.Furthermore, detailed functional assays were conducted to illustrate the underlying mechanism, which were lacking in the previous genetic studies on PIK3CG.As a lipid and protein kinase, PI3Kγ has been extensively studied in both cardiac cells and leukocytes. 38The role of kinase functions and kinase-independent functions of PI3Kγ in myocardium showed completely opposite effects. 23Numerous studies have demonstrated the protective role of kinase-independent scaffolding function of PI3Kγ in heart failure. 10,19,21In contrast, its kinase function appears to be detrimental, 21,25 leading to controversies in the field. 9,23Overall, PI3Kγ confers cardio-protection in response to various pathological stimuli.Importantly, our study revealed that individuals with homozygous mutation had higher PIK3CG expression in both protein levels of human heart and mRNA levels of peripheral blood lymphocytes.These individuals with homozygous mutation also exhibited a favourable prognosis for HF, aligning with the beneficial role of PI3Kγ observed in mice. 10rthermore, our study revealed that rs10215499-G allele destroyed the binding site of hsa-miR-133a-3p, resulting in increased transcription of PIK3CG.Consequently, this transcriptional upregulation contributes to a more favourable prognosis for heart failure (Figure 8).
Additionally, we observed no significant difference in the level of hsa-miR-133a-3p among heart tissues with different genotypes, with the heart exhibiting the highest level of miR-133a-3p.Our study indicated that hsa-miR-133a-3p could reduce the expression of PIK3CG and suggested a potential involvement of hsa-miR-133a-3p in cardiac pathophysiology, which needs further investigation in the future.
There are still some limitations in our study.First, we only focused on functional variants in PIK3CG gene.Other SNPs in linkage disequilibrium with rs10215499 may also affect the prognosis of HF and need further investigation.Second, there may be additional regulatory factors involved in the regulation of PIK3CG gene expression besides hsa-miR-133a-3p.Finally, the MAF of rs10215499 varies significantly among different racial groups, being highest in the Asian population and lowest in the European population.It is important to note that our study results may not be generalizable to other racial groups.
In conclusion, we have identified a common variant in PIK3CG gene significantly associated with the prognosis of HF in our discovery population, which was further validated in the replicated population.A series of functional assays indicated that rs10215499-G allele may destroy the binding site of hsa-miR-133a-3p, leading to increased transcription of PIK3CG and ultimately resulting in a better prognosis (Figure 8).Additionally, individuals with rs10215499-GG genotype exhibited higher mRNA and protein level of PIK3CG compared to those with AA or AG genotype, which supported the aforementioned functional investigations.Furthermore, overexpression of PIK3CG could protect AC16 from H/R-induced apoptosis, whereas silencing of PIK3CG had the opposite effect.Moreover,

F I G U R E 1
Functional analysis of rs1129293 synonymous variant.(A) Map of single-nucleotide polymorphisms (SNPs) in the promoter and exon of PIK3CG genotyped in 48 healthy individuals.(B, C) AC16 and HEK293T cells were transfected with expression plasmids engineered to harbour either rs1129293-C or -T allele.Empty vector pcDNA3.1 was served as control.The protein level of PIK3CG showed no difference between wild-type and mutant-type alleles.TA B L E 3 Haploblock structure of variants in PIK3CG gene with MAF >0.1 identified by Sanger sequencing.

F I G U R E 2 F I G U R E 3
PI3Kγ in myocardium, we attempted to investigate the association of variants in PIK3CG gene with the prognosis of HF.Interestingly, Effects of rs1129293 on the prognosis of HF patients.Cox proportional hazards models analysis showed that rs1129293 was associated with the prognosis of HF in the first cohort (A) replicated cohort (B) and combined cohort (C).In vivo PIK3CG transcription and translation differs by genotype.(A) Absolute PIK3CG mRNA levels (relative to housekeeping gene ACTB) were measured in total RNA preparations from lymphocytes and compared among different genotypes.(B) Western blot analysis of 17 human failing heart samples showed the presence of rs1129293-T allele increased protein expression of PIK3CG.*p < 0.05.

F I G U R E 5 F I G U R E 6
In vitro inhibitory effect of hsa-miR-133a-3p on PIK3CG expression.(A, B) AC16 and HEK293T cells were identified to be rs10215499-AA genotype by sanger sequencing.(C, D), hsa-miR-133a-3p could significantly reduce PIK3CG protein levels in both AC16 and HEK293T cells, which could be reversed by miR-133a-3p inhibitors.*p < 0.05.The expression level of hsa-miR-133a-3p.(A) hsa-miR-133a-3p level showed no difference in heart with different genotypes.(B) Comparison of hsa-miR-133a-3p expression level in different human tissues.NS, not significant.F I G U R E 7 Effect of PIK3CG on H/R-induced cardiomyocyte apoptosis in vitro.(A, B) PIK3CG-overexpression plasmid and PIK3CG-siRNAs showed significantly overexpression and inhibitory effects, respectively.(C) TUNEL assay detected TUNEL positive cells at 24 h after H/R treatment.*p < 0.05.
the high hazard ratios (HR = 1.96, 95% CI = 1.34-2.85;p = 0.0005) and high frequency of rs10215499 (MAF = 0.34) suggest that this SNP may account for a substantial proportion of poor prognosis of HF patients.Novel strategy through targeting PIK3CG is a promising way to decrease HF-associated mortality and improve the prognosis of HF.F I G U R E 8Role of hsa-miR-133a-3p in the Regulation of PIK3CG Transcription.The variant rs10215499, which is in strict LD with genotyped synonymous SNP rs1129293, is a A-to -G change in the 3′UTR of PIK3CG and located in the predicted binding site of hsa-miR-133a-3p.Rs10215499-G allele destroys the hsa-miR-133a-3p binding site and subsequently results in increased transcription and translation of PIK3CG, followed by reduced cardiomyocyte apoptosis under H/R condition, which ultimately favours better prognosis of HF.

2.6 | Protein extraction and immunoblots
to polyvinylidene difluoride membranes.Non-specific binding sites were blocked with 5% non-fat milk for 2 h at room temperature.

Table 4
, only rs1129293 demonstrated a significant association with the prognosis of HF in recessive model within the first cohort (p = 0.013).The association remained statistically significant even after adjustment for traditional risk factors including sex, age, hypertension, diabetes, hyperlipidemia, smoking state, and β-blocker use (p = 0.007, hazard ratio [HR] = 0.56) (Figure 2A).Subsequently, we attempt to confirm our discoveries in the replicated cohort.The results Abbreviation: MAF, minor allele frequency.a Base pair position is based on NCBI GRCH37.b

Haploblock structure Gene position a dbSNP ID b Gene region Maj > Min c MAF
Abbreviations: MAF, minor allele frequency; SNP, single nucleotide polymorphism.aBasepair position is based on NCBI GRCH37.bPolymorphism are numbered relative to transcription start site.cWith major allele given first, followed by minor allele.
Association of genetic polymorphisms in PIK3CG gene with HF prognosis.Note: Hazard ratio (HR) and 95% confidence intervals (95% CIs) were obtained using Cox regression, with and without adjustment for sex, age, hypertension, diabetes, hyperlipidemia, smoking status and β-blocker use.Bold and italic represents the statistical significance.
miR-133a-3p.The results demonstrated that the expression level of TA B L E 4 Abbreviations: MAF, minor allele frequency; SNP, single-nucleotide polymorphism.